8-OH-Ade is a modified nucleotide base resulting from single electron oxidation reactions. Steenken, “Purine bases, nucleosides and nucleotides: Aqueous solution redox chemistry and transformation of their radical cations è and OH Adducts,” Chem. Rev., Vol. 89, pp. 503-520 (1989). The presence of this oxidized base has been detected in biological specimens in a manner consistent with toxicant exposure and carcinogenesis. Malins et al., “The Etiology of Cancer: hydroxyl radical-induced DNA lesions in histologically normal livers of fish from a population with liver tumors,” Aquat. Toxicol., Vol. 20, pp. 123-130 (1991) and Malins et al., “The Etiology of Breast Cancer,” Cancer Vol. 71, No. 10, pp. 3036-3043 (1993). Previous methods of detecting and quantitating 8-OH-Ade have relied on analysis by high performance liquid chromatography-electrochemical detection (HPLC-ECD) (Shigenaga et al., “Urinary 8-hydroxy-2′-Deoxyguanosine as a Biological Marker of In Vivo Oxidative DNA Damage,” Proc. National. Acad. Sci. USA, Vol. 86, pp. 9697-9701 (1989)) and gas chromatography-mass-spectrometry with selected ion monitoring (GC-MS/SIM) (Malins et al. (1993)) procedures. Recently, assays utilizing polyclonal antibodies have also been used to detect 8-OH-Ade. West et al., “Radioimmunoassay of 7, 8-dihydro-8-oxoadenine (8-hydroxyadenine),” Int. J. Radiat. Biol., Vol. 42, No. 5, pp. 481-490 (1982). The present invention provides new and improved materials and methods for detecting and quantitating the 8-OH-Ade base structure in a biological specimen comprising a group of highly specific monoclonal antibodies against 8-OH-Ade which may be used in a variety of immunoassays.
Oxygen-free radicals are the primary mediators of cellular free-radical reactions. They are produced in normal or pathological cell metabolism, and as a result of exposure to a variety of exogenous sources of oxidative stress such as tobacco smoke, fatty acids in foods, iron and copper ions, and ethanol. Furthermore, ultraviolet light and ionizing radiation can stimulate the generation of oxygen-free radicals.
Oxygen-free radicals cause constant damage which the body's antioxidant defense systems usually repair so that a dynamic equilibrium is maintained. However, occasionally an overabundance of oxygen free radicals in the body occurs. Oxidative stress refers to the condition in which there is an overproduction of oxygen-free radicals or a deficiency in the antioxidant defense and repair mechanisms. Examples of short-term oxidative stress reactions include ischemia, reperfusion injury, acute inflammation and hyperoxia. Dreher et al., “Role of Oxygen Free Radicals in Cancer Development,” European Journal of Cancer, Vol. 32A(1), pp. 30-38 (1996).
While short-term oxidative stress does not generally result in severe or debilitating illness, chronic oxidative stress can result in oxidative damage to an organisms DNA, which in turn has been associated with a variety of diseases. It has been established that reactive oxygen species play a significant role in mutagenesis, carcinogenesis and tumor promotion. Bhimani et al., “Inhibition of Oxidative Stress in HeLa Cells by Chemopreventive Agents,” Cancer Research, Vol. 53, pp. 4528-4533 (1993). The dismutation of superoxide yields hydrogen peroxide, which is highly reactive in vivo. Hydrogen peroxide reacts with partially reduced metal ions to form the hydroxyl radical which can directly inflict DNA damage. Dreher et al. (1996). If hydroxyl radicals are generated close to DNA, they can attack the purine and pyrimidine bases, causing mutations. Halliwell, “Free Radicals, Antioxidants, and Human Disease: Curiosity, Cause, or Consequence?, The Lancet, Vol. 344, pp. 721-724 (1994). A discussion of the mechanisms of oxygen-free radical related mutagenesis resulting from DNA damage can be found in Dreher et al., Role of Oxygen-Free Radicals in Cancer Development, European Journal of Cancer, Vol. 32A, No. 1, pp. 30-38 (1996).
The involvement of reactive oxygen species in the development of cancer in humans is supported by the abundant presence of oxidative DNA modifications in cancer tissue. Loft et al., “Cancer Risk and Oxidative DNA Damage in Man,” J. Mol. Med., Vol.74, pp.297-312 (1996). For example, in breast cancer, base lesion concentrations have been found to be substantial. Base lesions previously reported include 8-OH-Ade, among others. Malins, et al. (1990). It is believed that these base lesions play a pivotal roll in oncogenesis and may further serve as early predictors of breast cancer risk, in addition to a variety of other cancers, due to their inherently mutagenic and carcinogenic effects. See Halliwell (1994) and Bhimani et al. (1993) and Dreher et al. (1996).
Reactive oxygen species have also been implicated in the etiology and pathophysiology of many other human diseases including cardiovascular disease, chronic inflammatory disease, neurodegenerative diseases, rheumatoid arthritis, systemic lupus, erythematosis and sickle cell anemia. Bhimani et al. (1993) and Halliwell (1994).
The etiology of these diseases and their progression, as well as toxicant exposure, can be studied by measuring levels of oxidized DNA bases in biological specimens. Levels of oxidized DNA bases, such as 8-OH-Ade, may also be used as a predictor of risk of disease, thereby allowing preventive intervention before the clinical disease develops. Strickland et al., “Methodologies for Measuring Carcinogen Adducts in Humans,” Cancer Epidemiology, Biomarkers and Prevention, Vol. 2, pp. 607-619 (1993).
One of the most abundant and most studied oxidative modifications of DNA-bases is the C-8 hydroxylation of guanine. Loft et al., “Cancer Risk and Oxidative DNA Damage in Man”, J. Mol. Med., Vol. 74, pp. 297-312 (1996). Other abundant oxidatively modified purines and pyrimidines include 8-oxoadenine, 2-hydroxyadenine, Fapy-A, 5-hydroxy cytosine, and thymine glycol, among others. Loft et al. (1996). Of interest to the present invention in the modified purine 8-OH-Ade.
The presence of oxidative damage in genomic and mitochondrial DNA obtained from biological specimens such as tissues and isolated cells has been studied by a variety of methods, including most commonly gas chromatography/mass spectroscopy with selective ion monitoring (GC/MS-SIM) and high-performance liquid chromatographic separation (HPLC) followed by detection by UV or electrochemistry. Four primary methods used for monitoring and quantifying oxidative DNA damage in biological specimens are discussed in detail in the review article by Strickland et al. (1993) and include immunoassay techniques, post-labeling, fluorescence spectroscopy and GC/MS.
In view of the great interest in DNA adducts, including 8-OH-Ade, efforts have been made to make antibodies sensitive to these oxidized nucleosides. The use of antibodies to detect DNA adducts began in the mid-1970s. Strickland et al. (1993). A variety of assays utilizing the antibodies have been developed, including the competitive radioimmunoassay, solid phase enzyme immunoassay, competitive solid phase enzyme immunoassay, ELISA, and immunoaffinity chromatography. Strickland et al. (1993). Monoclonal antibodies have been used to detect, for example, 1-methyladenosine and pseudouridine in urine (Matsuda et al., “An Immunohistochemical Analysis for Cancer of the Esophagus Using Monoclonal Antibodies specific for Modified Nucleosides,” Cancer, Vol. 72, pp. 3571-3578 (1993); various derivatives of guanosine and thymine (Adamkiewicz et al., “Monoclonal Antibody-Based Immunoanalytical Methods for Detection of Carcinogen-Modified DNA Components,” Arc Sc. Publ., Vol. 70, pp. 403-41 (1986); and 8-OH-guanine (Lee et al., “Identification of 8-Hydroxyguanine Glycosylase Activity in Mammalian Tissues Using 8-Hydroxyguanine Specific Monoclonal Antibody,” Biochemical and Biophysical Research Communications, Vol. 196, No. 3, pp. 1545-1551 (1993) and Yin et al., “Determination of 8-Hydroxyde-oxyguanosine by an Immunoaffinity Chromatography-Monoclonal Antibody-Base Elisa,” Free Rad. Biol. & Med., Vol. 18(6), pp. 1023-1032 (1995)). In the article “Radial Immunoassay of 7, 8-Dihydro-8 Oxoadenine (8-Hydroxyadenine),” West et al. (1982), the authors discuss a specific radioimmunoassay for 8 hydroxy-adenine, and its application in the study of irradiated adenine solutions as well as a preliminary measurements of the production of 8-hydroxyadenine, using polyclonal antibodies.
Although detection and quantitation of oxidized nucleoside basis by immunoassay is gaining popularity, the challenge is in producing a suitable antibody for a specific base. It is preferable to have a specific and highly sensitive antibody which exhibits little cross-reactivity with related molecules. Polyclonal antibodies lack specificity and assays using such antibodies lack sensitivity. Therefore, use of monoclonal antibodies are preferred. However, in practice, obtaining suitable monoclonal antibodies can be difficult. The technique of producing monoclonal antibodies by hybridoma technology is well known in the art. Nevertheless, the results obtained by this technique are unpredictable. Only by carrying out the process for making the monoclonal antibodies can the nature of the, monoclonal antibodies be determined and ascertained. To the bast of the inventors knowledge, no assay has been developed for the detection and quantitation of 8-OH-Ade in a biological specimen using monoclonal antibodies.
Therefore, what is needed in the art is a highly sensitive and specific assay for detecting the presence of and quantitating the amount of 8-OH-Ade present in a biological specimen.